Real complete three-dimensional reconstruction of Golgi-impregnated neurons by means of a confocal laser scanning microscope

Golgi-impregnated neurons of the human or animal central nervous system were studied with a confocal laser scanning microscope (CLSM). The scanning properties (optical sectioning of the specimen) offered by the CLSM and the capacity of metal granules to reflect the laser beam allow a three-dimensional reconstruction of the impregnated neurons. The volume scanned can be depicted in three different ways: (a) extended focus, i.e., a bidimensional image that contains information from all the optical sections, as if there were an extensive depth of focus; (b) a topographic representation in which the intensity of every pixel is proportional to the calculated z value (as a result, the closer the object section is to the surface, the greater the color intensity becomes); and (c) shadow representation, i.e., a pseudo-three-dimensional image. In addition, a true and complete three-dimensional reconstruction of neurons can be obtained using an extended RAM and quick elaboration (fast CPU) combined with the rotation of the reconstructed image in the different planes. High-magnification, high-numerical-aperture (NA) oil immersion objective lenses with reduced working distance may present some problems in the three-dimensional reconstruction of large neurons with extensive and spreading dendritic branches. This limitation may be overcome by using a low-magnification (10 x) oil immersion lens.